1. Field of the Invention
The present invention relates to a method of introducing at least one gene encoding a product into at least one cell of a mammalian host for use in treating the mammalian host. This method discloses employing vector molecules containing a gene encoding the product and infecting, for example, connective tissue cells of the mammalian host using the vector molecule. This invention provides both viral and non-viral methods of introducing at least one gene encoding a product into at least one cell of the mammalian host to treat the mammalian host.
More specifically, the present invention discloses ex vivo and in vivo techniques for delivery of DNA sequence of interest to the connective tissue cells of the mammalian host. The ex vivo technique involves prior removal and culture of target connective tissue cells, in vitro infection of the DNA sequence by vector or other delivery vehicle into the connective tissue cells, and transplantation of the modified connective tissue cells to the mammalian host such as of a target joint, so as to effect in vivo expression of the gene product of interest. As an alternative, non-connective tissue cells, such as hematopoietic progenitor cells, stromal cells, bone marrow cells, myoblasts, leukocytes or mature lymphoid or myeloid cells may be transfected in vitro, recovered and injected into the bone marrow or blood stream of the patient using techniques known to the skilled artisan. These cells can also be injected locally into the connective tissue.
The in vivo technique bypasses the requirement for in vitro culture of target cells, instead relying on direct transplantation of the DNA sequence by vector or other delivery vehicle to the target cells in vivo, thus effecting expression of the gene product of interest. For example, the gene encoding the product of interest can be introduced into liposomes and injected directly into the area of the joint, where the liposomes fuse with synovial cells, resulting in an in vivo gene transfer to synovial tissue. Alternatively, the gene encoding the product of interest can be introduced into the area of the joint as naked DNA. The naked DNA enters the synovial cell, resulting in an in vivo gene transfer to synovial tissue.
The present invention also relates to a method for producing an animal model for the study of connective tissue pathologies and systemic indices of inflammation.
2. Brief Description of the Related Art
Arthritis involves inflammation of a joint that is usually accompanied by pain and frequent changes in the structure of the joint. Arthritis may result from or be associated with a number of conditions including infection, immunological disturbances, trauma and degenerative joint diseases, such as osteoarthritis. The biochemistry of cartilage degradation in joints and cellular changes have received considerable investigation.
In a healthy joint, cells in cartilage (chondrocytes) and the surrounding synovium (synoviocytes) are in a resting state. In this resting state, these cells secrete basal levels of prostaglandins, cytokines and various proteinases, such as collagenase, gelatinase and stromelysin, with the ability to degrade cartilage. During the development of an arthritic condition, these cells become activated. In the activated state, synoviocytes and chondrocytes synthesize and secrete large amounts of prostaglandins, cytokines and proteinases.
In efforts to identify pathophysiologically relevant cell activators, it has been known that the cytokine interleukin-1 activates chondrocytes and synoviocytes and induces cartilage breakdown in vitro and in vivo. Additionally interleukin-1 is a growth factor for synoviocytes and promotes their synthesis of matrix, two properties suggesting the involvement of interleukin-1 in the synovial hypertrophy that accompanies arthritis. In contrast, interleukin-1 inhibits cartilaginous matrix synthesis by chondrocytes, thereby suppressing repair of cartilage. Interleukin-1 also induces bone resorption and thus may account for the loss of bone density seen in rheumatoid arthritis. Interleukin-1 is inflammatory, serves as a growth factor for lymphocytes, is a chemotactic factor and a possible activator of polymorphonculear leukocytes (PMNs). When present in a sufficient concentration, interleukin-1 may cause fever, muscle wasting and sleepiness.
The major source of interleukin-1 in the joint is the synovium. Interleukin-1 is secreted by the resident synoviocytes, which are joined under inflammatory conditions by macrophages and other white blood cells.
Much attention has been devoted to the development of a class of agents identified as the "Non-Steroidal Anti-Inflammatory Drugs" (hereinafter "NSAIDs"). The NSAIDs inhibit cartilage synthesis and repair and control inflammation. The mechanism of action of the NSAIDs appears to be associated principally with the inhibition of prostaglandin synthesis in body tissues. Most of this development has involved the synthesis of better inhibitors of cyclo-oxygenase, a key enzyme that catalyzes the formation of prostaglandin precursors (endoperoxides) from arachidonic acid. The anti-inflammatory effect of the NSAIDs is thought to be due in part to inhibition of prostaglandin synthesis and release during inflammation. Prostaglandins are also believed to play a role in modulating the rate and extent of leukocyte infiltration during inflammation. The NSAIDs include drugs such as acetylsalicylic acid (aspirin), fenoprofen calcium (Nalfon.RTM. Pulvules.RTM., Dista Products Company), ibuprofen (Motrin.RTM., The Upjohn Company), and indomethacin (Indocin.RTM., Merck and Company, Inc.).
Therapeutic intervention in arthritis is hindered by the inability to target drugs, such as the NSAIDs, to specific areas within a mammalian host, such as a joint. Traditional routes of drug delivery, such as oral, intravenous or intramuscular administration, depend upon vascular perfusion of the synovium to carry the drug to the joint. This is inefficient because transynovial transfer of small molecules from the synovial capillaries to the joint space occurs generally by passive diffusion. This diffusion is less efficient with increased size of the target molecule. Thus, the access of large drug molecules, for example, proteins, to the joint space is substantially restricted. Intra-articular injection of drugs circumvents those limitations; however, the half-life of drugs administered intraarticularly is generally short. Another disadvantage of intra-articular injection of drugs is that frequent repeated injections are necessary to obtain acceptable drug levels at the joint spaces for treating a chronic condition, such as arthritis. Because therapeutic agents heretofore could not be selectively targeted to joints, it was necessary to expose the mammalian host to systemically high concentrations of drugs in order to achieve a sustained, intra-articular therapeutic dose. Exposure of non-target organs in this manner exacerbated the tendency of anti-arthritis drugs to produce serious side effects, such as gastrointestinal upset and changes in the hematological, cardiovascular, hepatic and renal systems of the mammalian host.
It has been shown that genetic material can be introduced into mammalian cells by chemical or biological means. Moreover, the introduced genetic material can be expressed so that high levels of a specific protein can be synthesized by the host cell. Cells retaining the introduced genetic material may include an antibiotic resistance gene thus providing a selectable marker for preferential growth of the transduced cell in the presence of the corresponding antibiotic. Chemical compounds for inhibiting the production of interleukin-1 are also known.
U.S. Pat. No. 4,778,806 discloses a method of inhibiting the production of interleukin-1 by monocytes and/or macrophages in a human by administering through the parenteral route a 2-2'-[1,3-propan-2-onediyl-bis(thio)]bis-1 H-imidazole or a pharmaceutically acceptable salt thereof. This patent discloses a chemical compound for inhibiting the production of interleukin-1. By contrast, in one embodiment of the present invention, gene therapy is employed that is capable of binding to and neutralizing interleukin-1.
U.S. Pat. No. 4,780,470 discloses a method of inhibiting the production of interleukin-1 by monocytes in a human by administering a 4,5-diaryl-2 (substituted) imidazole. This patent also discloses a chemical compound for inhibiting the production of interleukin-1.
U.S. Pat. No. 4,794,114 discloses a method of inhibiting the 5-lipoxygenase pathway in a human by administering a diaryl-substituted imidazole fused to a thiazole, pyrrolidine or piperidine ring or a pharmaceutically acceptable salt thereof. This patent also discloses a chemical compound for inhibiting the production of interleukin-1.
U.S. Pat. No. 4,870,101 discloses a method for inhibiting the release of interleukin-1 and for alleviating interleukin-1 mediated conditions by administering an effective amount of a pharmaceutically acceptable anti-oxidant compound such as disulfiram, tetrakis [3-(2,6-di-tert-butyl-4-hydroxyphenyl) propionyloxy methyl] methane or 2,4-di-isobutyl-6-(N,N-dimethylamino methyl)-phenol. This patent discloses a chemical compound for inhibiting the release of interleukin-1.
U.S. Pat. No. 4,816,436 discloses a process for the use of interleukin-1 as an anti-arthritic agent. This patent states that interleukin-1, in association with a pharmaceutical carrier, may be administered by intra-articular injection for the treatment of arthritis or inflammation. In contrast, the present invention discloses a method of using and preparing a gene that is capable of binding to and neutralizing interleukin-1 as a method of resisting arthritis.
U.S. Pat. No. 4,935,343 discloses an immunoassay method for the detection of interleukin-1 beta that employs a monoclonal antibody that binds to interleukin-1 beta but does not bind to interleukin-1 alpha. This patent discloses that the monoclonal antibody binds to interleukin-1 beta and blocks the binding of interleukin-1 beta to interleukin-1 receptors, and thus blocking the biological activity of interleukin-1 beta. The monoclonal antibody disclosed in this patent may be obtained by production of an immunogen through genetic engineering using recombinant DNA technology. The immunogen is injected into a mouse and there-after spleen cells of the mouse are immortalized by fusing the spleen cells with myeloma cells. The resulting cells include the hybrid continuous cell lines (hybridomas) that may be later screened for monoclonal antibodies. This patent states that the monoclonal antibodies of the invention may be used therapeutically, such as for example, in the immunization of a patient, or the monoclonal antibodies may be bound to a toxin to form an immunotoxin or to a radioactive material or drug to form a radio pharmaceutical or pharmaceutical.
U.S. Pat. No. 4,766,069 discloses a recombinant DNA cloning vehicle having a DNA sequence comprising the human interleukin-1 gene DNA sequence. This patent provides a process for preparing human interleukin-1 beta, and recovering the human interleukin-1 beta. This patent discloses use of interleukin-1 as an immunological reagent in humans because of its ability to stimulate T-cells and B-cells and increase immunoglobulin synthesis.
U.S. Pat. No. 4,396,601 discloses a method for providing mammalian hosts with additional genetic capability. This patent provides that host cells capable of regeneration are removed from the host and treated with genetic material including at least one marker which allows for selective advantage for the host cells in which the genetic material is capable of expression and replication. This patent states that the modified host cells are then returned to the host under regenerative conditions.
U.S. Pat. No. 4,968,607 discloses a DNA sequence encoding a mammalian interleukin-1 receptor protein which exhibits interleukin-1 binding activity.
U.S. Pat. No. 5,081,228 discloses a DNA sequence encoding both the murine and human interleukin-1 receptor. This patent also provides a process for the in vitro expression of said DNA sequences.
U.S. Pat. No. 5,081,812 discloses a substantially pure preparation of the human interleukin-1 receptor protein.
Patent Application WO9634955 discloses a method of treating an arthritic condition using recombinantly modified articular chondrocytes.
U.S. Pat. No. 5,643,752 discloses a host cell transformed with an expression vector containing nucleic acid amino acids 30-224 of the TIMP-4 polypeptide.
Patent Application WO9723639 discloses expression vectors containing DNA encoding a protein having the formula A-X-B, where A and B are subunits of a dimeric protein or are each a biologically active protein; X is a linker polypeptide. Transformed hosts containing the vectors are also disclosed. The method reportedly can be used for the production of interleukin-12 using DNA coding for the 40 Kd and 35 Kd subunits of IL-12, joined by a suitable linker.
Patent Application WO9700958 discloses an isolated nucleic acid encoding pCL13, a member of TGF-.beta. family member, having immunosuppressant, cell differentiation promoting and anti-proliferative activities.
In spite of these disclosures, there remains a very real and substantial need for a method for introducing at least one gene encoding a product of interest into at least one cell of a mammalian host in vitro, or alternatively in vivo, for use in treating the mammalian host. Further, there is a need for a process wherein a gene encoding a soluble interleukin-1 receptor is used to resist the deleterious pathological changes associated with arthritis. There is also a need to utilize one or more DNA sequences for delivery to and expression of a protein or protein fragment with a target host connective tissue cell, such as a synovial cell, or non-connective tissue cell so as to effect a treatment of various joint pathologies and concomitant systemic indices of inflammation. A further need exists to provide an animal model for the study of joint pathologies.